Frosty pod rot in cacao (Moniliophthora roreri) is the main limitation on the production of cacao (Theobroma cacao) in Mexico. A sustainable alternative for the control of the disease is the use of the Trichoderma mushroom. The objective of this study was to select isolates that are native to Trichoderma with the best antagonist and physiological in vitro characteristics for the control of M. roreri. For this, 50 isolates of Trichoderma obtained in the cacao agroecosystem were characterized. Mycelial growth and the production of conidia at 25, 30 and 35 °C were considered the physiological variables. Mycoparasitism, antibiosis and potential antagonism were the antagonist variables. Significant differences (P = 0.0001) were found in all evaluated variables. The interval of the optimal temperature for mycelial growth and the production of conidia was 25 to 30 °C. Mycoparasitism varied between 0 and 100 %, and only the isolates of six species showed this characteristic. Antibiosis varied between 6.8 and 55.5 % and potential antagonism varied from 3.4 to 69.0 %. Trichoderma virens (TTC017) and T. harzianum (TTC090, TTC039, TTC073) showed the best potential in vitro biocontrol, so they are promising strains for future investigations on biological control of cacao moniliasis.

Cultural practices have been the most commonly used method for the fight against frosty pod
rot in cacao (Soberanis et al., 1999). The
use of fungicides has been a scarcely used practice, due to the erratic strategies
of fungicide evaluations and to the fluctuating price of cacao (Bateman et al., 2005). Programs
for the development of resistant genetic material have also been established (Phillips-Mora, Arciniegas-Leal, Mata-Quiros, &
Motomajor-Arias, 2012); however, notable progress has not been obtained
in the commercial use of clones with resistance to the disease. Krauss and Soberanis (2001) mention that
biocontrol offers some potentials in the sustainable handling of the frosty pod rot
through the use of antagonists. The authors reported that the species of the
Trichoderma genus showed control on M. roreri.

In the search of biological control agents, one of the basic strategies should be the initial exploration of the natural native enemies (Vázquez, Matienzo, Veitía, & Alfonso, 2008). Based on this reasoning, in the state of Tabasco, Mexico, there are 50 isolates of Trichoderma obtained from the T. cacao rhizosphere which have been grouped into nine species and may be evaluated for the control of M. roreri. Due to this, the objective of this study was to select isolates native to Trichoderma with the best in vitro antagonist and physiological characteristics for the control of M. roreri.

Materials and methods

Trichoderma isolates

Fifty isolates of Trichoderma grouped in nine species were characterized in the present work (Table 1). These isolates were obtained from the T. cacao rhizosphere as a part of a diversity study of Trichoderma in the cacao agroecosystem in the state of Tabasco, Mexico, which were confirmed in kind through morphology and ITS sequences (Torres-de la Cruz et al., 2015). Currently, these isolates are a part of the Trichoderma collection of the División Académica de Ciencias Biológicas, Universidad Juárez Autónoma de Tabasco. The M. roreri isolate was provided by the Laboratorio de Fitopatología of the Colegio de Postgraduados Campus Tabasco, previously identified through morphology and ITS sequences (GenBank access number: GU108605). All isolates were preserved and multiplied in the middle of the Potato Dextrose Agar (PDA) culture.

zThe isolates were identified with the letters TTC (Trichoderma Tabasco
Cacao) followed by the number of isolates.

Mycoparasitism of Trichoderma on M. roreri

The mycoparasitic ability of the Trichoderma isolates on M.
roreri was evaluated according to the pre-colonized Petri dish
method (Evans, Holmes, & Thomas,
2003). A fragment of 5 mm in diameter was obtained from M.
roreri colonies of 10 days of age and was placed near the edge of a
Petri dish with 9 cm in diameter with half PDA. The Petri dishes with an
inoculated medium were incubated during 37 days at 25 ± 1 °C in the
dark. Subsequently, a 0.5 x 2.5 cm fragment of the
Trichoderma inoculum, obtained from a 4 day old colony, was
planted on the opposite side of the M. roreri inoculum. The
dishes pre-colonized by M. roreri and inoculated with
Trichoderma were incubated during 15 days under the same
conditions indicated for pre-colonization. Five repetitions of the isolate and
five of the control treatment were established. The control treatment comprised
pre-colonized dishes of M. roreri without the inoculation of
Trichoderma.

After the incubation, 10 samples of 0.5 mm in diameter were extracted initiating the inoculum of M. roreri in direction to the inoculum of Trichoderma. The samples were planted in Petri dishes with PDA mediums and were incubated at 25 ± 1 °C in the dark. The samples were observed during 7 days in order to detect the presence of the mycoparasite (Trichoderma) or phytopathogen (M. roreri) and evaluate the percentage of mycoparasitism with the following formula:

PP = (CT x 100) / N

where:

PP =

Parasitism (%)

CT =

Samples with Trichoderma growth

N =

Number of samples extracted from each replica

Antibiosis of Trichoderma on M. roreri

The antibiosis of Trichoderma isolates on M. roreri was
evaluated on cultures coupled according to Holmes, Schroers, Thomas, Evans, and Samuels (2004). Antibiosis was
calculated by the percentage of radial growth reduction of M.
roreri. For this, a fragment of 10 day old M.
roreri of 5 mm in diameter was placed on a Petri dish with PDA
medium. The inoculated dishes were incubated in the dark during 7 days at 25 ± 1
°C in order to establish the colony. Subsequently, the colony of M.
roreri was controlled with the mycoparasite, for which a 5 mm
fragment of 4 day old Trichoderma was planted on the opposite
side of the M. roreri. All dishes were incubated at 25 ± 1 °C
in the dark; five repetitions per isolate and control treatment were
established. The control treatment comprised colonies of M.
roreri without confrontation with Trichoderma. The
radial growth was recorded daily until one of the isolates had mycelial contact
with M. roreri. The percentage of mycelial growth inhibition
was determined with the Abbott formula
(1925):

PA = [(CR - CRT) / CR] x 100

where:

PA =

Antibiosis (%)

CR =

Radial growth of M. roreri without Trichoderma (mm)

CRT =

Radial growth of M. roreri in the presence of Trichoderma (mm)

Potential antagonism of Trichoderma on M. roreri

The potential antagonism of the Trichoderma isolates on M. roreri was obtained through the formula:

AP = (MP + PA) / 2

where:

MP =

Mycoparasitism of isolates of Trichoderma on M. roreri (%)

PA =

Antibiosis of isolates of Trichoderma on M. roreri (%)

Mycelial growth of Trichoderma

Mycelial growth was evaluated according to Dimbi, Maniania,
Lux, and Mueke (2004). A fragment of 5 mm in diameter was obtained
from the margins of 4 days old Trichoderma colonies and was
placed in the middle of a Petri dish with a PDA medium. The boxes were incubated
at 25, 30 and 35 °C (the three temperatures with ± 1 °C) with a photoperiod of
12 h in the light and 12 h in the dark; five repetitions per isolate and per
temperature were established. The rate of mycelial growth was recorded each 12
h; the test was finalized when one of the colonies filled the Petri dish. The
measurements of the last record were used in the statistical analysis. Radial
growth (r) was transformed into the growth area (A, cm2) with the formula A =
πr2. The corresponding area to each mm of radial growth was expressed in
percentages. Thus, the effect of the temperature on the mycelial growth was
evaluated through the inhibition percentage and the increase of the area, when
increasing the temperature from 25 to 30 °C and from 25 to 35 °C.

Production of Trichoderma conidia

From the 4 days old Trichoderma cultures, a fragment of 5 mm in diameter was planted in Petri dishes with PDA medium. The inoculated fragment was incubated at 25, 30 and 35 °C (the three temperatures with ± 1 °C), with a photoperiod of 12 h in the light and 12 h in the dark during 16 days. Five repetitions per isolate and per temperature were established. The conidia were cultivated from the culture surface and the count was done in a Neubauer chamber (Hausser Scientific, USA). The concentration of conidia·mL-1 was estimated through the following formula:

C = (Cc) (4 x 106) (Fd / 80)

where:

C =

Concentration (conidia·mL-1)

Cc =

Average conidia counted in the Neubauer chamber

Fd =

Dilution factor

The effect of the temperature on the production of conidia was evaluated in the same manner
as indicated for mycelial growth.

Statistical analysis

The information on mycoparasitism, antibiosis, potential antagonism, mycelial growth and production of conidia were analyzed under a completely random design. Mycoparasitism, antibiosis and potential antagonism consisted of 50 treatments (each one of the evaluated isolates). Mycelial growth and the production of conidia comprised 40 treatments, as the isolates that did not show mycoparasitism and that had the lowest AP values were discarded. Prior to the analysis, the information on mycoparasitism, antibiosis and potential antagonism were transformed to the arc sine of the square root of the proportion, and the information on mycelial growth and production of conidia was transformed to log (x + 1). The information was subjected to an ANOVA and a separation of Tukey means test (P < 0.05) through SAS® (Statistical Analysis System [SAS], 1998).

Results and discussion

Mycoparasitism of Trichoderma on M. roreri

Table 2 presents the percentage of
mycoparasitism of the 50 isolates native of Trichoderma
evaluated on M. roreri. The isolates showed significant
differences (P = 0.0001) and the percentage varied between 0 and 100 %. New
isolates reached 100 % mycoparasitism at 15 days of incubation and 10 isolates
did not show this characteristic. The isolates with mycoparasitism on M.
roreri belong to the T. harzianum (Rifai),
T. virens (Mill, Giddens & Foster) Arx, T.
spirale (Bissett), T. brevicompactum (Kraus,
Kubicek & Gams), T. koningiopsis (Samuels, Suárez &
Evans) and T. asperellum (Samuels, Lieckf & Nirenberg)
species. It could be hypothesized that these species produce a matrix of enzymes
that allow parasitism of M. roreri. In this regard,
mycoparasitism of T. harzianum, T virens,
T. asperellum and T. koningiopsis isolates
on M. roreri has been reported by Evans et al. (2003) and Krauss et al. (2006), while mycoparasitism
of T. brevicompactum and T. spirale was
documented for the first time. In this study, the parasitism of isolates of one
same species varied, which is consistent with Hoyos-Carvajal, Duque, and Orduz (2008), who indicated that isolates
of the same species can act in different ways.

± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

The isolates that did not show parasitism on M. roreri correspond to
T. pleuroticola (Yu & Park), T.
longibrachiatum (Rifai) and T. reesei (Simmons).
The foregoing coincides with Garcia-Simoes,
Tauk-Tornisielo, Rocha-Niella, and Tapia-Tapia (2012), who evaluated
this species without finding parasitism on M. perniciosa
(Stahel) Aime and Phillips-Mora, a species that is related to M.
roreri. Trichoderma pleuroticola has also been
reported as a pathogen of edible mushrooms (Sobieralski et al., 2012). The absence of mycoparasitism of these
species suggests that M. roreri is not in the group of hosts.

Trichoderma antibiosis on M. roreri

Table 3 presents the results of the
antibiosis test of Trichoderma on M. roreri.
All isolates showed antibiosis with significant differences between them (P =
0.0001). The percentage of antibiosis fluctuated from 6.8 to 55.5 %; the highest
values (40 to 55.5%) were obtained with some isolates of T.
asperellum (TCC051 and TCC024) T. koningiopsis
(TCC084, TCC020 and TCC063), T. pleuroticola (TCC002),
T. virens (TCC021) and T. harzianum
(TCC056, TCC077 and TCC115). The antibiotic action of the T.
harzianum isolates has also been described on the mycelial growth
of M. roreri and M. perniciosa (Bailey et al., 2008).

Table 3 Antibiosis of 50 native species to Trichoderma on
Moniliophthora roreri.

Isolate

Antibiosis (%)

Isolate

Antibiosis (%)

TTC051

55.5 ± 4.0 a

TTC016

30.9 ± 3.0 ghijklmno

TTC084

52.8 ± 2.7 ab

TTC026

29.7 ± 4.0 hijklmnop

TTC020

48.9 ± 5.0 abc

TTC058

28.5 ± 3.7 hijklmnopq

TTC002

46.0 ± 3.7 abcd

TTC076

28.5 ± 4.8 Ijklmnopq

TTC021

44.3 ± 3.8 bcde

TTC062

27.8 ± 1.5 Ijklmnopq

TTC024

43.2 ± 4.9 bcdef

TTC109

27.3 ± 7.2 jklmnopq

TTC056

41.2 ± 2.7 cdefg

TTC003

26.6 ± 3.2 klmnopq

TTC077

41.0 ± 4.0 cdefg

TTC113

25.8 ± 4.2mnopq

TTC115

40.5 ± 0.6 cdefg

TTC037

25.1 ± 4.7 mnopqr

TTC063

40.2 ± 5.6 cdefg

TTC054

24.7 ± 3.8 nopqrs

TTC027

38.7 ± 1.2 cdefgh

TTC015

23.0 ± 1.5 opqrst

TTC017

37.9 ± 3.7 defghi

TTC045

21.1 ± 2.0 pqrstu

TTC102

37.5 ± 6.7 defghi

TTC081

20.3 ± 2.6 pqrstuv

TTC085

37.1 ± 2.3 defghij

TTC047

19.9 ± 2.6 qrstuvw

TTC039

36.1 ± 4.0 defghijk

TTC104

19.8 ± 6.3 qrstuvw

TTC035

35.6 ± 5.0 defghijkl

TTC014

16.8 ± 3.7 rstuvw

TTC001

35.3 ± 3.3 efghijkl

TTC031

16.2 ± 2.6 stuvw

TTC004

34.9 ± 1.4 efghijklm

TTC086

16.0 ± 1.4 stuvw

TTC088

34.7± 3.9 efghijklm

TTC093

15.9 ± 4.0 tuvw

TTC073

33.9 ± 3.5 efghijklmn

TTC023

15.6 ± 2.9 tuvw

TTC007

33.4 ± 2.9 fghijklmn

TTC059

14.8 ± 3.9 tuvw

TTC028

32.4 ± 2.1 ghijklmno

TTC101

14.5 ± 3.4 uvw

TTC090

32.4 ± 3.7 ghijklmno

TTC008

13.1 ± 4.1 vwx

TTC050

31.7 ± 2.1

TTC032

12.3 ± 1.5 wx

TTC009

31.5 ± 2.6

TTC100

6.8 ± 2.5 x

± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

The isolates of T. longibrachiatum, T. reesei and
T. pleuroticola showed antibiosis on M.
roreri (Table 3), even
though they did not present mycoparasitism (Table 2). The presence of antibiosis without mycoparasitism suggest
that the isolates have metabolites with antifungal activity, or that they do not
allow the growth of the pathogen, due to the space and nutrient competition
mechanisms (García-Simoes et al., 2012).

Potential antagonism of Trichoderma on M. roreri

Table 4 shows that there were significant
differences (P = 0.0001) in the potential antagonism of
Trichoderma on M. roreri; the percentage
varied between 6.8 and 68.8 %. To evaluate the antagonism potential, such as the
sum of mycoparasitism and antibiosis acting synergistically, it was observed
that some isolates of T. virens (TTC017, TTC058 and TTC015),
T. harzianum (TTC062 and TTC090) and T.
spirale (TTC004) showed the highest values, while the lowest
percentages were obtained with the isolates that did not show mycoparasitism.
According to Monte (2001), the
combination of parasitism and antibiosis could result in significant
antagonistic levels.

± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

Mycelial growth of Trichoderma

The statistical analysis showed significant differences (P = 0.0001) in the mycelial growth
of the Trichoderma isolates in each temperature that was
assessed. The results are presented in Table
5. At 25 °C, mycelial growth varied between 21 and 40 mm. The TTC063
and TTC084 strains of T. koningiopsis, and TTC021 and TCC017 of
T. virens obtained the highest growth at 60 h. At 30 °C,
the mycelial growth fluctuated between 18.3 and 40 mm; the TCC021, TCC017,
TCC102 and TCC104 strains of T. virens, TCC084 of T.
koningiopsis, and TCC100, TCC073 and TCC090 of T.
harzianum expressed the highest growth. When going from 25 to 30
°C, 30 out of 40 isolates increased their growth and three maintained it
(TTC021, TTC084 and TTC017). Figure 1 shows
the graphic comparison of the changes that happened on the mycelial growth of
the Trichoderma isolates with temperature changes. At 35 °C,
mycelial growth fluctuated from 2.1 to 29 mm and the TCC017 strain (T.
virens) presented the highest growth. At this temperature, all the
isolates decreased the mycelial growth with respect to the one obtained at 25
and 30 °C; the inhibition fluctuated from 23 to 99.5 % (Figure 1). All isolates grew in the three increased temperatures; however, the range of favorable
temperatures for 82 % of the isolates was 25 to 30 °C. In this regard, Jalil, Norero, and Apablaza (1997) reported
that the optimal temperature for T. harzianum is 27 °C. The
notable growth of all strains in an interval between 25 and 35 °C suggests the
adaptability of the high temperatures where cacao is grown.

± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

Figure 1 Inhibition changes or increase of mycelial growth of native isolates to
Trichoderma when surpassing 25 to 30 °C and 25
to 35 °C.

Production of Trichoderma conidia

The statistical analysis showed significant differences (P = 0.0001) in the production of
conidia of the Trichoderma isolates in each evaluated
temperature (Table 6). At 25 °C, the
production fluctuated from 1.4 x 107 to 3.1 x 109 conidia·mL-1. The strains with
the highest production were TTC063 and TTC051 of the T.
koningiopsis and T. asperellum species,
respectively. At 30 °C, the production of conidia varied between 0.05 x 108 and
2.6 x 109 conidia·mL-1; the TTC102 and TTC017 strains of T.
virens, and TTC115 of T. harzianum presented the
highest production. At this temperature, 65 % of the isolates increased the
production of conidia in relation to the production obtained at 25 °C (Figure 2). At 35 °C, the production of
conidia varied between 0 and 3.9 x 108 conidia·mL-1. The TTC084 isolates
(T. koningiopsis), TTC104 and TTC016 (T.
virens) showed the highest production. At this temperature, 97.5 %
of the isolates decreased the production of conidia, with respect to the
obtained conidia at 25 and 30 °C; six isolates showed total inhibition (Figure 2). The inhibition fluctuated from
12.3 to 100 %. Only the TTC104 (T. virens) isolate increased
the production of conidia, in regards to the values obtained at 25 and 30 °C.

Table 6 Conidia production of 40 isolates native to Trichoderma at 25, 30 and
35 °C.

Isolate

Production (conidia·mL-1)

25 °C

30 °C

35 °C

TTC063

3.1 x 109 ± 1.6 x 108

a

1.3 x 109 ± 3.3 x 108

efgh

1.2 x 106 ± 2.1 x 105

lm

TTC051

2.1 x 109 ± 2.4 x 108

b

1.1 x 109 ± 1.6 x 108

fghij

2.7 x 106 ± 1.0 x 105

I

TTC001

1.9 x 109 ± 3.5 x 107

bc

1.8 x 109 ± 5.4 x 107

bc

4.9 x 106 ± 1.5 x 105

h

TTC035

1.8 x 109 ± 1.1 x 108

bc

1.2 x 109 ± 1.0 x 108

efghi

1.3 x 107 ± 2.7 x 106

g

TTC102

1.8 x 109 ± 1.4 x 108

bc

2.6 x 109 ± 2.4 x 108

a

4.2 x 107 ± 3.5 x 106

e

TTC084

1.6 x 109 ± 1.2 x 108

bcd

8.9 x 108 ± 3.9 x 106

ijklm

3.9 x 108 ± 2.6 x 107

a

TTC002

1.6 x 109 ± 2.1 x 108

cd

1.3 x 109 ± 2.2 x 108

efgh

2.1 x 106 ± 5.1 x 105

Ij

TTC020

1.4 x 109 ± 3.6 x 107

de

1.2 x 109 ± 5.2 x 107

efghij

1.7 x 106 ± 1.0 x 105

jk

TTC015

1.3 x 109 ± 2.1 x 108

def

1.2 x 109 ± 1.1 x 108

efghi

2.5 x 105 ± 0

pq

TTC073

1.3 x 109 ± 2.3 x 108

defg

1.1 x 109 ± 1.3 x 108

fghij

6.4 x 106 ± 3.5 x 105

h

TTC024

1.3 x 109 ± 2.8 x 107

defg

1.7 x 109 ± 7.4 x 107

bcd

1.3 x 106 ± 3.0 x 105

kl

TTC037

1.2 x 109 ± 1.3 x 108

efg

1.5 x 109 ± 1.4 x 108

cdef

8.6 x 107 ± 1.7 x 106

cd

TTC045

1.1 x 109 ± 1.2 x 108

efgh

2.8 x 108 ± 2.8 x 107

opqr

6.6 x 107 ± 1.0 x 106

d

TTC021

1.0 x 109 ± 8.7 x 107

fghi

1.7 x 108 ± 1.0 x 107

qr

1.3 x 106 ± 1.7 x 105

klm

TTC016

1.0 x 109 ± 6.8 x 107

fghij

1.4 x 109 ± 1.4 x 108

defg

1.6 x 108 ± 1.5 x 107

b

TTC090

1.0 x 109 ± 8.9 x 107

fghij

1.5 x 109 ± 1.9 x 108

bcde

1.4 x 106 ± 1.9 x 105

kl

TTC115

9.9 x 108 ± 4.1 x 107

ghijk

2.3 x 109 ± 6.8 x 107

a

1.0 x 106 ± 0

m

TTC032

8.4 x 108 ± 3.7 x 107

hijkl

9.7 x 108 ± 8.2 x 107

hijkl

7.4 x 107 ± 4.4 x 106

d

TTC017

8.2 x 108 ± 2.7 x 107

ijklm

1.9 x 109 ± 2.1 x 108

b

2.6 x 107 ± 3.0 x 106

f

TTC109

7.9 x 108 ± 2.3 x 107

jklmn

1.4 x 109 ± 2.7 x 107

defg

1.0 x 108 ± 2.7 x 106

c

TTC058

7.6 x 108 ± 4.4 x 107

klmn

1.0 x 109 ± 1.5 x 108

ghijk

2.8 x 105 ± 3.7 x 104

nop

TCC023

7.2 x 108 ± 7.8 x 106

lmno

1.3 x 109 ± 8.5 x 107

efgh

3.5 x 105 ± 1.7 x 104

n

TTC085

7.1 x 108 ± 1.6 x 107

lmno

8.1 x 108 ± 1.0 x 108

jklm

0 ± 0

r

TTC077

6.6 x 108 ± 1.5 x 107

lmno

1.7 x 109 ± 3.4 x 108

bcd

4.1 x 107 ± 2.7 x 106

e

TTC062

6.3 x 108 ± 3.6 x 107

mno

9.0 x 108 ± 1.0 x 108

ijklm

2.6 x 105 ± 2.3 x 104

nopq

TTC039

6.3 x 108 ± 2.1 x 107

mno

1.5 x 109 ± 8.8 x 107

cdef

1.4 x 107 ± 5.1 x 105

g

TTC031

6.3 x 108 ± 2.6 x 107

mno

7.1 x 108 ± 4.1 x 107

klmn

2.2 x 106 ± 2.1 x 105

Ij

TTC014

6.3 x 108 ± 1.2 x 107

no

1.4 x 109 ± 1.1 x 108

defg

2.5 x 105 ± 1.1 x 104

opq

TTC047

6.2 x 108 ± 2.3 x 107

no

9.0 x 108 ± 5.0 x 107

ijklm

0

r

TTC086

5.8 x 108 ± 2.6 x 107

op

8.2 x 108 ± 8.0 x 107

jklm

2.5 x 105 ± 0

pq

TTC009

4.7 x 108 ± 3.1 x 107

pq

8.0 x 108 ± 8.6 x 107

jklm

1.3 x 107 ± 3.8 x 106

g

TTC100

4.7 x 108 ± 4.1 x 107

pq

1.5 x 109 ± 2.6 x 108

cdef

1.3 x 107 ± 3.4 x 106

g

TTC004

4.3 x 108 ± 1.9 x 106

qr

5.6 x 108 ± 2.7 x 107

mnop

0

r

TTC059

4.3 x 108 ± 2.7 x 107

qr

4.1 x 108 ± 5.6 x 107

nopq

3.4 x 105 ± 2.5 x 104

no

TTC050

3.6 x 10 8 ± 5.0 x 107

r

6.0 x 108 ± 1.1 x 108

lmno

0

r

TTC054

3.4 x 108 ± 2.4 x 107

r

3.8 x 108 ± 2.3 x 107

nopq

0

r

TTC076

3.3 x 108 ± 1.4 x 107

r

2.0 x 108 ± 2.6 x 107

pqr

2.7 x 105 ± 2.9 x 104

nopq

TTC104

1.8 x 107 ± 3.7 x 106

s

0.05 x 108 ±4.8 x 103

r

1.9 x 108 ± 1.7 x 107

b

TTC056

1.5 x 107 ± 3.5 x 106

s

1.1 x 108 ± 2.2 x 107

qr

2.0 x 105 ± 0

q

TTC093

1.4 x 107 ± 2.8 x 106

s

1.1 x 108 ± 1.4 x 107

qr

0

r

± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

Figure 2 Inhibition changes or increase in the production of isolate conidia native to
Trichoderma when surpassing 25 to 30 °C and 25
to 35 °C.

Given that the spores are the active structures of the mushrooms that are the agents for
biocontrol, the production of conidia is an important characteristic for the
selection of promissory isolates (Vélez-Arango,
Estrada-Valencia, González-García, Valderrama- Fonseca, &
Bustillo-Pardey, 2001). In this regard, 85 % of the isolates produced
conidia in the interval from 25 to 35 °C (Table
6), which could be explained by the tropical origin of the isolates;
however, the range of the favorable temperature for production was 25 to 30 °C.
The isolates with the most production of conidia in the range of evaluated
temperatures were: TTC063 (T. koningiopsis); TTC051, TTC001,
TTC035 (T. asperellum); TTC102 (T. virens);
and TTC115 (T. harzianum), with a superior production of 1 x
109 conidia·mL-1.

Conclusions

The results show that in the cacao agroecosystem, six out of nine existing species of
Trichoderma present antagonistic capacities on M.
roreri. According to the data presented in this work, the
Trichoderma spp. isolates showed an intraspecific variability
in regards to parasitism, antibiosis, mycelial growth and sporulation. The rank of
favorable temperature for the native isolates of Trichoderma was 25
to 30 °C. Based on the evaluated characteristics, the promissory isolates for the
biological control of M. roreri are: TTC017 of T.
virens, and TTC090, TTC039 and TTC073 of T. harzianum.
The variability of the isolates in the assessed characteristics demonstrates the
importance of in vitro characterization and manifests the potential of the native
species of Trichoderma for the development of bio fungicides on
M. roreri. Future studies will have to be implemented for the
evaluation of the isolates selected under field conditions.

Acknowledgements

This work was financed by the Consejo Nacional de Ciencia y Tecnología (CONACYT) and the Programa de Mejoramiento del Profesorado (PROMEP) of the Secretaría de Educación Pública.